Targeting dendritic cells for priming cellular immune responses

JOURNAL OF MOLECULAR RECOGNITIONJ. Mol. Recognit. 2003; 16: 299–317DOI:10.1002/jmr.650

Review

Targeting dendritic cells for priming cellularimmune responses

Peter Gogolak, Bence Rethi, Gyorgy Hajas and Eva Rajnavolgyi*Institute of Immunology, Faculty of Medicine, University of Debrecen, 98 Nagyerdei Blvd, Debrecen H-4012, Hungary

The cardinal role of dendritic cells (DC) in priming adaptive immunity and in orchestrating immuneresponses against all classes of pathogens and also against tumors is well established. Their unique potentialboth to maintain self-tolerance and to initiate protective immune responses against foreign and/ordangerous structures is based on the functional diversity and flexibility of these cells. Tissue DC liningantigenic portals such as mucosal surfaces and the skin are specialized to take up a wide array of compoundsincluding proteins, lipids, carbohydrates, glycoproteins, glycolipids and oligonucleotides, particles carryingsuch structures and apoptotic or necrotic cells. This process is facilitated by specialized receptors with highendocytic capacity, which provides potential targets for delivering designed molecules. The best route fortargeting B- and/or T cell epitopes, however, is still the subject of intense investigation. Immature DC, whichreside in various tissues, can be activated by pathogens, stress and inflammation or modified metabolicproducts, which induce mobilization of cells to draining lymph nodes where they act as highly potentprofessional antigen presenting cells. This is brought about by the ability to present their accumulatedintracellular content for both CD4þ helper (Th) and CD8þ cytotoxic/cytolytic T lymphocytes (Tc/CTL).Engulfed proteins are processed intracellularly and their peptide fragments are transported to the cellsurface in the context of major histocompatibility complex encoded class I and II molecules for presentationto Th cells and CTLs, respectively. The T cell priming capacity of DC, however, depends not only on antigenpresentation but also on other features of DC.

Human monocyte-derived DC provide an excellent tool to study the internalizing, antigen-presenting andT cell-activating functions of DC at their immature and activated differentiation states. These biologicalactivities of DC, however, are highly dependent on their migratory potential from the peripheral non-lymphoid tissues to the lymph nodes, on the expression of adhesion molecules, which support the interactionof DC with T lymphocytes, and the cytokines secreted by DC, which polarize immune responses to Th1-mediated cellular or Th2-mediated antibody responses. These results altogether demonstrate that mono-cyte-derived DC are useful candidates for in vitro or in vivo targeting of antigens to induce efficient adaptiveimmune responses against pathogens and also against tumors. Copyright # 2003 John Wiley & Sons, Ltd.

Keywords: dendritic cell; antigen presentation; targeting; T lymphocyte; cellular immune response; virus- and tumor-specific immunity; vaccination

Received 15 April 2003; revised 12 May 2003; accepted 19 May 2003

THE GENERAL FEATURES OFDENDRITIC CELLS

This article gives an overview of the complexity and flex-ibility of dendritic cell (DC) functions and discusses the

possibilities of targeting DC activities to induce protectiveanti-viral and therapeutical anti-tumor immunity. DC to-gether with monocytes and macrophages belong to themononuclear phagocytic system and act as non-specificeffector cells against microorganisms and tumors. They arealso involved in the uptake, processing and presentation ofantigens and thus act as professional antigen-presenting cells(APC). Unique properties characteristic for DC render themmuch more potent in priming immune responses than mono-cytes or macrophages (Van Voorhis et al., 1983; Banchereauand Steinman, 1998; Guermonprez et al., 2002).

DC represent a rare but heterogeneous population ofcells, which develop from bone marrow-derived CD34þhematopoietic stem cells and differentiate into circulatingprecursors generating different lineages (Plate 1; Pulendranet al., 1997; Liu, 2001; Ardavin et al., 2001; Banchereauet al., 2000). The two major routes of DC differentiationresult in the myeloid and lymphoid (plasmocytoid) DC

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: E. Rajnavolgyi, 98 Nagyerdei Blvd, Debrecen H-4012,

Hungary.

E-mail: evaraj@jaguar.dote.hu

Abbreviations used: ACAMP, apoptotic cell associated membrane protein; APC,

antigen-presenting cell; CR, complement receptor; CTL, cytolytic T lymphocyte;

DC, dendritic cell; FcR, Fc receptor; FDC, follicular dendritic cells; FISH,

fluorescent in situ hybridization; HMGB1, high mobility group 1 protein; HSP,

heat shock protein; LC, Langerhans cell; MR, mannose receptor; MRP, multidrug

resistance associated protein; MVB, multivesicular body; NK, natural killer cell;

PPR, pattern recognition receptor; PS-R, phosphatidyl serine receptor; SAGE,

serial analysis of gene expression; SR, scavenger receptor; TCR, T cell receptor;

Th1, helper type 1, inflammatory T cell; Th2, helper type 2, anti-inflammatory T

cell; TSLP, thymic stromal lymphopoietin; TSP, thrombospondin.

subpopulations with distinct tissue distribution and func-tions. Myeloid DC include Langerhans cells (LC), whichreside in the epidermis of the skin as well as in the mucosalepithelium. Interstitial DC are present in all non-lymphoidtissues and develop from blood precursor cells upon migra-tion through endothelial barriers. Blood monocytes are alsoable to differentiate to monocyte-derived DC, which resem-ble interstitial DC in their functions. The origin of lymphoidDC detectable in the thymus, blood and tonsils is stillcontroversial (Plate 1; Olweus et al., 1997).

The biological activity of DC is highly dependent on theirorigin and differentiation state. Distinct DC subsets anddifferentially activated DC may be involved in opposingmechanisms such as inducing tolerance or immune re-sponses, activating inflammatory (Th1), anti-inflammatory(Th2) or regulatory T-lymphocytes (Rajnavolgyi and Lanyi,2003; Bach, 2003). When activated properly, myeloid DCinduce the activation of Th1 cells and thus become potentinitiators of anti-viral and anti-tumor immune responses.Uptake of high amounts of antigen, however, is not suffi-cient to prime inflammatory responses. To accomplish thisfunction DC also require ‘danger signals’, which can beprovided by the antigen itself, such as virus-infected cell ornecrotic tumor cells, or by signals mediated by other cells ofthe innate immune system such as macrophages or naturalkiller (NK) cells. The terminal maturation of DC is inducedby the contact with T-lymphocytes in the special micro-environment of lymphoid tissues, which allows the intimateinteraction of DC and T cells by an immunological synapse(Plate 2; van der Merwe, 2002).

Dendritic cells reside in non-lymphoid tissues in theirantigen capturing immature state (iDC) and upon activationevolve to mature professional APC (mDC), which are ableto prime T-cell responses. During this process DC convertantigens to immunogens or tolerogens, express adhesionand co-stimulatory molecules to promote cell-to-cellinteractions, release soluble molecules such as cytokines,chemokines and enzymes, which regulate cellular comm-unication and mobilization (Guermonprez et al., 2002;Banchereau and Steinman, 1998). iDC localize in highnumbers close to antigenic portals and are characterizedby extensive antigen uptake mediated by pinocytosis,macropinocytosis or phagocytosis (Flores-Romo, 2001).These cells are equipped with a wide array of internalizingreceptors capable of binding proteins, carbohydrates, lipidsor nucleotides (Plate 3). Another important function of someof these receptors is to receive and transmit activation andmaturation signals for further differentiation. Thus iDC actas specialized sensors of tissue-associated changes andtransfer this information to T-lymphocytes (Reis e Sousa,2001). Transition from the immature to the mature differ-entiation states can be induced by various stimuli and isaccompanied by phenotypic and functional changes as wellas by tissue redistribution. Activation of iDC can be inducedby inflammatory or pathogenic products derived from mi-crobes or from damaged tissues (Viney, 2001). These stimuliinduce the mobilization of DC from non-lymphoid tissues todraining lymphoid organs via afferent lymphatic vessels(Plate 4). Migration is accompanied by down-regulation ofphagocytosis and receptor-mediated internalization, re-dis-tribution of MHC molecules from intracellular vesicles tothe cell surface, re-organization of the cytoskeleton,

formation of dendrites, increased cell surface expressionof co-stimulatory molecules and chemokine receptors, andsecretion of various cytokines and chemokines. Homing ofactivated DC occurs primarily in the T cell-rich areas oflymph nodes where they interact with naive T-lymphocytes,which express specific clonally distributed receptors forrecognizing antigenic peptides presented by MHC mole-cules on the DC surface. The immunological synapseformed between DC and T cells is stabilized by multipleco-stimulatory and adhesion molecules, while the specifi-city of the signaling complex is maintained by the MHC-peptide-TCR interaction (Plate 2). The expression pattern ofco-stimulatory molecules and cytokine receptors on DC andthe combination of released cytokines direct the polarizationof CD4þ helper T-lymphocytes either to Th1 and/or to Th2effectors. Priming of CD8þ cytotoxic T-lymphocytes issupported by Th1 cells, whereas Th2 cells provide helpfor B cells and facilitate antibody production, isotypeswitch, and affinity maturation of memory B-cells (Plate5; Maldonado-Lopez and Moser, 2001). Effector T-lympho-cytes leave the lymphoid tissues and migrate back to theinflammed tissue, where they recognize virus infected ormalignant cells. As a result of effector T-cell functions therecognized cells are killed by cytotoxic T-lymphocyteswithout damage to normal tissue cells.

Owing to their complex biological activities DC play apivotal role in (a) activating antigen-specific T-lymphocytesand priming cellular immune responses, (b) polarizinghelper T-lymphocytes to Th1 and/or to Th2 effector cells,(c) priming CD8þ T-lymphocytes and supporting differen-tiation of cytotoxic effectors, (d) maintaining immunologi-cal memory at the level of both T- and B-lymphocytes, and(e) inducing regulatory T-lymphocytes and maintainingperipheral immunological tolerance.

TARGETING DENDRITIC CELLFUNCTIONS

Large body of experimental data revealed that DC representa highly flexible cell population (Gluckman et al., 2002),which can be targeted at multiple levels both in vitro and invivo. Availability of genome sequences of various pathogensand gene array analysis of different tumors opened up newpossibilities for the identification of target antigens by‘reverse vaccination’ strategies. These approaches are basedon the computer-assisted analysis of genomic sequences andthe selection of candidate antigens (Rappuoli, 2001). Theprogress in identifying novel antigens relevant for subunitvaccines against pathogens and tumors posed the require-ment of more efficient antigen delivery systems and adju-vants than those offered by traditional methods. Helper Tlymphocyte-assisted antibody responses are efficient againstcertain pathogens but do not eliminate most cancer or virus-infected cell. Anti-viral and anti-tumor vaccines shouldhave the capability to elicit MHC class I-restricted cytotoxicT cell responses induced by endogenous viral or tumorantigens or by highly potent professional APC such as DC.

Although DC are considered as professional scavengersand phagocytes, antigen uptake facilitating the activation ofboth types of T lymphocytes, especially loading of MHC

300 P. GOGOLAK ET AL.

Copyright # 2003 John Wiley & Sons, Ltd. J. Mol. Recognit. 2003; 16: 299–317

Plate 1. Differentiation and localization of various dendritic cell subsets. Dendritic cells differentiate from CD34þ hemopoieticstem cells along different pathways (based on Liu et al., 2001). Myeloid blood precursors differentiate to monocyte-derived,interstitial and Langerhans type DC, which in their immature state reside in non-lymphoid tissues, skin andmucosal epithelium.Monocyte-derived and interstitial DC phagocytose bacteria, lymphoid DC secrete IFN� upon viral infection as effector cells ofinnate immunity. Activation results in differentiation to mature DC, which act as professional APC and activate cellular immuneresponses. In the absence of antigens and danger signals DC are involved in the maintenance of peripheral tolerance.

Plate 2. The immunological synapse. The interaction of activated DC and T lymphocytes occurs in peripheral lymphoid organs.The direct contact of the two cells is stabilized by the interaction of adhesionmolecules, whereas the specificity of T cell activationis ensured by theMHC–peptide–T cell receptor complex. Besides TCR-mediated signaling, other receptor–ligand interactions alsomediate intracellular signals and thus act as co-stimulatory molecules.

TARGETING DENDRITIC CELLS

Copyright # 2003 John Wiley & Sons, Ltd. J. Mol. Recognit. 2003; 16

Plate 3. Receptors and functions of immature tissue-resident dendritic cells. Immature dendritic cells expressa wide array of internalizing receptors involved in the uptake of various compounds. The engulfed material isstored and transported in intracellular vesicular compartments. Processing of internalized antigens andloading of antigenic fragments onto MHC class I and class II molecules is induced by DC activation.

Plate 4. Migration and homing of dendritic cells. Immature tissue resident DC constitutively take up exogenoussoluble or particulate antigens. Danger signals delivered by pathogens or inflammation induce DC activationand mobilization. Activated DCmigrate to the draining lymph nodes via afferent lymphatics and home to T cellareas. Naive T lymphocytes recirculate in blood and enter lymph nodes via endothelium. Interaction ofactivated DC with specific T lymphocytes occurs in the special microenvironment of peripheral lymphoidorgans and results in T cell activation and differentiation. Effector and memory T lymphocytes leave thelymphoid tissue and migrate back to the site of infection or inflammation.

P. GOGOLAK ET AL.

Plate 5. The antigen presenting and T cell activating function of mature dendritic cells. Mature dendritic cells are professionalantigen presenting cells, which are able to activate both CD4þ helper and CD8þ cytotoxic T lymphocytes. Activation of Th1cells by mature myeloid DC results in the secretion of inflammatory cytokines and in the expression of activation molecules.The collaboration of Th1 cells with DC results in the condition of DC for priming CD8þ T cells and for facilitating thedifferentiation of cytolytic effector T cells. Helper T lymphocytes also support B cell differentiation and antibody production.

TARGETING DENDRITIC CELLS

class I molecules by exogenous antigens, is still a challengefor dendritic cell-based anti-viral or anti-tumor vaccination.Polarization of DC towards more potent anti-viral and anti-tumor responses (maturation of DC1 instead of DC2) is stillthe major goal of vaccine design. Rapid mobilization andefficient recruitment of DC to the site of infection or oftumor growth also emerges as an important requirement forinducing protective immune responses. The high engulfingcapacity, the regulated antigen processing and presentingfunctions, and the extreme sensitivity of DC to microenvir-onmental changes together with their high functional flex-ibility offer multiple possibilities for targeting DC activity.The novel approaches explored recently for vaccinationeither utilize cell therapy with antigen-loaded DC stimu-lated ex vivo or targeting antigens to DC in vivo.

The major function of tissue DC is to sample variouscompounds by fluid phase uptake, receptor-mediated inter-nalization and engulfment of particles (Plate 3). Within theAPC the exogenous molecules are handled in endo-/lyso-somal compartments where partial or complete enzymaticdegradation occurs. Protein fragments, generated in thespecial MHC class II-rich compartments of DC, are loadedto newly synthesized MHC class II molecules and stabilizetheir conformation. This interaction facilitates the transpor-tation of the correctly folded molecules together with thecaptured peptide to the cell surface (Simon et al., 2000). Incontrast to other cells DC are able to utilize exogenousantigens for loading MHC class I molecules and thusmediate the priming of CD8þ T lymphocytes, a mechanismreferred as ‘cross priming’ (Plate 5; Shen et al., 1997).

The identification of pathogenic and tumor-associatedantigens in various types of tumors allowed the developmentof cancer vaccines based on defined antigenic proteins orpeptides (Rosenberg, 1995; Boon and Old, 1997). Under invitro or in vivo conditions these antigenic moieties can bedelivered as naked plasmid DNA, recombinant viral orbacterial vectors, total RNA, purified recombinant proteinsor synthetic peptides. The traditional methods for deliveringantigens, however, are in many cases inefficient and/orinadequate. The efficacy of delivering antigens into DCcan be increased by (a) generating sufficient amounts ofantigenic peptides to achieve high intracellular antigenconcentrations in the right intracellular compartments, (b)targeting tumor antigens to the most efficient APC, (c)loading both MHC class I and class II membrane proteins,and (d) providing an appropriate ‘danger signal’ for theactivation of APC.

In clinical settings the efficacy of vaccines, their ability toconfer long-lasting immunity and/or to boost already exist-ing responses, safety, costs, and simplicity of production anddelivery are important further issues. Newly developed cellseparation techniques and the wide range of recombinantcytokines have opened up new avenues in the developmentof vaccination strategies against pathogens and tumors.

Targeting dendritic cell differentiation

In vitro manipulated purified DC do not require selectivetargeting of antigens and the proper triggering for T cellactivation can readily be achieved in vitro. However, thegrowth and functional activity of professional APC can be

modulated by various cytokines both in vivo and in vitro.Blood monocytes can be differentiated to macrophages byculturing them on hydrophobic surfaces in the presence ofhuman serum (Zou and Tam, 2002; Andreesen et al., 1983)or together with M-CSF that acts as a survival factor formonocytes and macrophages (Brugger et al., 1991). GM-CSF is widely used to generate DC in combination withother cytokines. IL-4 was shown to inhibit macrophagedifferentiation and induce the generation of monocyte-derived DC. Separation of these two lineages has beenquestioned recently due to the overlapping physiologicalfunctions of iDC and macrophages and to the ability ofmacrophages to differentiate into iDC and vice versa (Humeet al., 2002). In vitro generation of DC in the presence ofGM-CSF and IL-4 belongs to the most extensively char-acterized and utilized protocols in DC-related experimenta-tion and also in DC-based immunotherapy approaches,however, numerous other cytokines were shown to substi-tute IL-4 in DC-differentiation (Zou and Tam, 2002). Someof these cytokines share receptor subunits with IL-4, such asIL-2 and IL-7 (common �-chain), IL-13 and IL-15 (common�- and �-chains, respectively). All of these cytokines wereshown to induce DC differentiation. The generated cellsexpressed co-stimulatory molecules, and activated allo-geneic T lymphocytes, although their phenotype and someof their biological activities were different. These includethe expression of a CD21-like molecule selectively inducedby IL-7 (Takahashi et al., 1997) and the Langerhanscell-like features obtained in the presence of IL-15(Mohamadzadeh et al., 2001). Other cytokines, not relatedto IL-4, were also shown to promote the in vitro differenta-tion of monocyte-derived DC. TGF�, together with GM-CSF alone or with GM-CSFþ IL-4 skews the DC phenotypetowards Langerhans cells (Guironnet et al., 2002). Type Iinterferons in combination with GM-CSF were also shownto promote monocyte-derived DC differentation and ma-turation (Paquette et al., 1998). These data demonstrate thatDC represent a highly flexible cell population and thatcytokines can be used to modulate DC functions. Furthercharacterization of DC differentiated in the presence ofvarious cytokines may uncover novel functional character-istics and enhance the clinical utility of these cells (Nestleet al., 2001).

The therapeutical potential of DC-inducing cytokineswas verified both in animal models in vivo and in humanstudies using tumor cell vaccines in combination with GM-CSF (Hill et al., 2002; Salgia et al., 2003; Scheibenbogenet al., 2003). Flt-3 ligand was identified as a highly activecytokine for promoting DC differentiation from CD34þhemopoietic stem cells (Pulendran et al., 1997). Injection ofFlt-3 ligand to mice or to healthy individuals elicits apronounced increase in the numbers of both myeloid andplasmocytoid DC precursors in peripheral blood (Plate 1;Morse et al., 2000). DC were targeted in vivo by plasmidDNA, which contained genes encoding for the tumor anti-gen together with GM-CSF or monocyte chemotactic pro-tein-3 (MCP-3; Biragyn et al., 1999; Syrengelas et al.,1996). In situ loading of DC with tumor antigen and Flt-3ligand resulted in long-term anti-tumor immunity in mice(Merad et al., 2002). Recent results also indicate that bothacute and chronic myeloid leukemia cells (AML and CML,respectively) can be converted to potent, dendritic cell-like

TARGETING DENDRITIC CELLS 301

APC in the presence of various cytokines (Choudhury et al.,1999; Paquette et al., 2002). A possible mechanism of thebeneficial effect of IFN� treatment of CML patients may bethe forced differentiation of leukemic cells to cells with DCcharacteristics. These modified DC-like cells efficientlypresented peptides generated from the endogeneously ex-pressed tumor antigens and elicited leukemia-specific cel-lular immune responses.

Cytokines can potentially be combined with any antigendelivery systems to improve the immune response qualita-tively or quantitatively. In addition to co-administration ofcytokines and antigens, the regulated release of cytokinescan be achieved by various delivery systems similar to thoseused for antigen loading.

Targeting antigen uptake by dendritic cells

Viral or tumor antigens can be taken up by DC as solubleproteins secreted or shed from live tumor cells. The mostefficient pathway of viral and tumor antigen loading to DC,however, seems to be the uptake of apoptotic or necrotictumor cells (Larsson et al., 2001). The novel antigendelivery systems for isolated DC are focused to achievethe efficient loading of both MHC class I and II molecules.Multiple re-administration of in vitro loaded and properlyconditioned DC is a promising new approach of individualimmune therapy. However, these methods are labor-inten-sive, expensive and require special sterile laboratory condi-tions. Therefore, the development of new antigen deliverysystems applicable for the in vivo loading of MHC class Iand class II molecules via the exogenous route is of specialinterest (Rea et al., 2001). The most promising site of in vivoadministration is the skin in view of the high potency ofresident epidermal LC in antigen uptake. Despite their low(1%) frequency, these cells account for 25% of the availableepidermal surface area (Abbas et al., 2000). This may offeran extremely efficient route for targeting antigens to skin-resident LC.

Uptake of soluble molecules. Immature DC take up var-ious soluble molecules by fluid phase pinocytosis or byreceptor-mediated internalization. A wide array of interna-lizing receptors, involved in efficient uptake to endo-/lyso-somal compartments, have been identified (Plate 3). Thesemolecules belong to the large family of pattern recognitionreceptors (PRR) distributed on cells of the innate immunesystem. C-type (calcium dependent) lectins represent themost prevalent PRR expressed on DC (Table 1). These type Imembrane receptors are characterized by carbohydraterecognition domains (CRD), which bind mannose or galac-tose side chains of glycoproteins followed by internalizationin an ATP-dependent manner. DC-SIGN and mannosereceptors (MR) are expressed by LC and DC at the samesite in the body; others are restricted to various DC subsets.Expression of MR and other members of this family isdown-regulated upon DC maturation. The natural ligandsof DC-SIGN are lentiviruses, Ebola virus and Mycobacter-ium tuberculosis, but it can also bind soluble ligands(Tailleux et al., 2003). Since DC-SIGN and DEC-205 aredendritic cell-specific lectins, they are promising candidatesfor targeting antigens to DC (Hawiger et al., 2001; Engeringet al., 2002).

The major limitation of peptide-based immunization isthe short half-life and the low immunogenicity of peptides.Polycationic peptides reduce the repulsion of negativelycharged cell membranes and thus facilitate uptake. Thisapproach has been widely used to enhance the transport ofproteins into cells among them professional APC (Buschleet al., 1997). Subcutaneous administration of tumor antigen-derived-peptides in combination with cationic poly-aminoacids, such as poly-L-arginine or poly-L-lysine, has beenshown to enhance the efficacy of cell-free vaccines (Schmidtet al., 1997). Combination of polycationic peptides and thefusion peptide of influenza hemagglutinin-2 (HA2) se-quence was also used to load DC and was shown to elicitcytotoxic T cell responses against model and tumor anti-gens. Inclusion of the fusion peptide, which is able to disruptthe endosomal membrane and to facilitate release to the

Table 1. C-type lectins identified in dendritic cells

Name Number of CRDa Ligand Expression Function Antigen

Type IDEC-205 10 IDCb MDCc # Recycling to late Uptake

CD205 Thymic epithelial endo/lysosome Clathrin Tyr motifMannose 8/2 functional IDC MDC # Recycling to

receptor Single terminal mannose Macrophage endosome ClathrinCD206 of pathogens Tyr motif

Type IILangerin 1 High mannose Skin epidermal Birbeck granules Uptake

CD207 oligosaccharide glycoprotein LCd

DC-SIGN 1 High mannose DC Skin dermis Adhesion ICAM-3/2 T-cell interactionCD209 glycoproteins M. tuberculosis Mucosal LPe Internalization to Uptake M. tuberculosis

HIV gp120 Tonsil T area late endo/lysosome inhibitionClathrin Tyr motif

Dectin-1 DC Macrophage ITAM PresentationDCIR Glycosylated IDC ITIM Uptake

ligands MDC #a CRD, carbohydrate binding domain. b IDC, immature dendritic cell. c MDC, mature dendritic cell. d Langerhans cell. e Lamina propria. # , down-regulated.

cytosol, resulted in 20 times higher CTL responses ascompared with the effect of the cationic peptide itself(Laus et al., 2000). Intradermal injection of tumor antigenswas also shown to confer preventive and therapeutic anti-tumor immune responses. The in vivo effect of the vaccinewas attributed to the rapid extracellular transport, augmen-ted loading of APC and helper T cell-independent activationof CD8þ effector CTL cells in the lymph nodes (Luhrs et al.,2002).

Uptake of particles. DC do not take up viable cells butingest modified or apoptotic cells. Erythrocytes can bemodified by biotinylation, which allows the high-affinityattachment of any biotinylated protein by avidin bridge(Magnani et al., 1994). Antigens bound to the surface ofmicroparticles were shown to improve antigen presentationdespite the lower phagocytic activity of DC compared tothat of macrophages (Shen et al., 1997). Polycationicmicroparticles have been shown to greatly enhance phago-cytosis by DC (Thiele et al., 2001). Hydrophylic surfacesare able to decrease, while lipophilic materials or ligandswhich bind to specific receptors are able to increase phago-cytosis (Torche et al., 2000). However, various proteinspresent in the serum or in the interstitial fluid can—depending on their individual characteristics—interferewith binding to proteins by competition for free bindingsites. Human serum albumin and IgA inhibit phagocytosis,whereas �2-human serum glycoprotein (�2GP), expressedalso in DC, has high opsonic activity (Thiele et al., 2003).�2GP was also proposed to act as a homing molecule and asa receptor of an unknown ligand (Dziegielewska et al.,1996).

Biodegradable polylactide-co-glycololide (PLG) micro-spheres were used for delivering and for the controlledrelease of antigens and cytokines encapsulated or coupledcovalently or noncovalently to particles (Rafferty et al.,1996). These particles were able to induce humoral immuneresponses against model antigens and the size of particles inthe range of 1–10 mm was shown to be critical for efficientphagocytosis by DC. These microspheres delivered antigenefficiently to the class I presentation pathway of macro-phages and DC via a TAP-dependent mechanism. Lowantigen dose was required for the induction of cytotoxicT-lymphocytes demonstrated both in vitro and in vivo(Raychaudhuri and Rock, 1998). Using a melanoma-asso-ciated peptide this delivery system resulted in protectiveanti-tumor immunity.

Uptake of nucleotides. Instead of loading DC with recom-binant proteins or peptides, DC were successfully trans-fected with total RNA isolated from tumor cells (Nair et al.,1998; Zhang et al., 1999). Despite their high endocytic andphagocytic activity, DC are refractory to nonviral transfec-tion. Viral or bacterial vectors, which invade the cytoplasmof cells, have been used to introduce antigens into profes-sional APC. Vectors that have been used for DC transduc-tion include vaccinia, canary pox, adenovirus, Listeriamonocytogenes, Salmonella, and Mycobacterium bovis ba-cillus Calmette-Guerin (Raychaudhuri and Rock, 1998).

Transduction of DC with bacterial or viral expressionconstructs for various tumor antigens is a promising method

for antigen loading, since it does not require the productionof recombinant proteins and allows the introduction ofmultiple epitopes. In addition to these advantages thisantigen delivery system ensures the endogenous source ofviral or tumor antigens and thus the efficient loading of bothMHC class I and class II molecules. It also allows theactivation of CD4þ and CD8þ T lymphocytes, a crucialrequirement for eliciting protective immune responses(Plate 5; Kaplan et al., 1999; Arthur et al., 1997; Schnellet al., 2000). Depending on the type of viral vector, thisapproach also allows the simultaneous delivery of both theantigen and the danger signal by the vector itself. DNAencoding various pathogen-derived or tumor-associatedantigens engineered into viral or bacterial vectors, however,may have adverse effects. Some of these vectors are highlyimmunogenic and their delivery by DC, as the most potentnatural adjuvant, induces neutralizing vector-specific anti-bodies or vector-specific effector T cells, which rendervaccination ineffective. For example adenovirus vectorsefficiently transduce DC, but the excess of highly immuno-genic viral antigens and the large virus load required forefficient transfection results in antigenic competition andsuppresses the T-cell stimulatory capacity of DC (Yanget al., 1995; Jonuleit et al., 2000).

Unwanted activation of DC upon gene therapy ap-proaches is a major limitation of long-term expression ofgenes introduced to differentiated cells. Gene therapies fortargeting monogenic disorders not only deliver self proteinsto which tolerance has not been developed, but providedanger signals for DC. This is mediated by the vector, whichmay attract cells of innate immunity by the DNA itself or byendogenous cellular signals. Cell death and the uptake ofapoptotic cells can cause inflammation and increased anti-gen presentation. As a result the immune system recognizesthe vector as foreign before the correcting gene product andeliminates the transduced cells (Brown and Lillicrap, 2002).

Another approach to gene delivery is offered by DNAvaccination, which involves the injection of plasmid expres-sion vectors, which can be acquired, transcribed and trans-lated by host cells. New nonviral systems have been alsodeveloped for the delivery of plasmid DNA by using a novelcationic peptide, CL22. This cationic peptide was used tocondense plasmid DNA and neutralize its negative charges.Immunization of mice with this vaccine resulted in en-hanced anti-tumor responses as compared to peptide-pulsedDC (Irvine et al., 2000). In another system a plasmid DNAwith the inserted beta-galactosidase gene was coated topolycationic nanoparticles engineered from warm oil-to-water microemulsion. Mannan was used for topical in vivotargeting of this model antigen to the MR of LC by coatingthe nanoparticles together with the entrapped dioleoylphophatidylethanolamine (DOPE; Cui and Mumper,2002). Primary immunization with this genetic vaccineresulted in a Th1 type proliferative T cell response andantibody production.

Uptake of opsonized antigens. Binding and internalizationof opsonized antigens by antibodies and/or by complementfragments to phagocytic cells is one of the most importanteffector functions of humoral immune responses. Cell sur-face receptors, which are involved in the uptake of opso-nized antigens are summarized in Table 2.

Antigens complexed with IgG type antibodies bind to andare taken up by various Fc receptors (FcR). Fc�RI/CD64,Fc�RII/CD32 and Fc�RIII/CD16 are expressed on DC andmacrophages. The expression of CD16 and CD64 definesdifferent monocyte subpopulations and DC precursors withdifferent functional properties (Sanchez-Torres et al., 2001;Almeida et al., 2001; Grage-Griebenow et al., 2000, 2001;Siedlar et al., 2000; Schakel et al., 1999).

The high-affinity Fc�RI is expressed exclusively onmyeloid cells among them DC. Cross-linking of Fc�RI orFc�RII but not Fc�RIII activates NF-�B, but still results inreduced IL-12 production and reduced antigen presentingfunction (Drechsler et al., 2002). Internalization of antigen-IgG complexes via Fc�RII (CD32) in human DC enhancedthe efficacy of antigen presentation 100-fold as comparedwith free antigen (Larsson et al., 1997). Fc�R-mediateddelivery of antigens to DC by internalization of immunecomplexes was shown to result in efficient peptide loadingof MHC class II molecules and to promote class I-restrictedcross-presentation at very low antigen concentration(Regnault et al., 1999). In myeloid cells, targeting exogen-ous antigens to Fc�RI by an antibody-based expressionvector resulted in cross presentation by MHC class I

molecules (Wallace et al., 2001). The contribution of IgGimmune complexes to the efficacy of active and passiveimmune therapy against melanoma was demonstrated andindicated the preferential role of macrophages in the effectorphase (Clynes et al., 1998). As revealed by myeloma cells,which were coated with anti-syndecan-1 antibody, this me-chanism could promote cross-presentation of cellular anti-gens and thus enhance the efficacy of therapeuticalmonoclonal antibody-based therapies. In this setting cross-presentation was inhibited by pretreatment of DCs withFc�R-blocking antibodies (Dhodapkar et al., 2002). Target-ing with IgG type antibodies, however, can result in opposingeffects due to the different intracellular signaling sequencesof various Fc�R. Using human tumor xenografts both theactivation and the inhibitory effect of different Fc�R wasdemonstrated (Clynes et al., 2000). The Fc�RIIB receptorcontains an inhibitory ITIM motif, which is able to interferewith the stimulatory effect of other Fc�R upon co-cross-linking by immune complexes (reviewed in Unkeless and Jin,1997). The anti-tumor activity of trastuzumab (Herceptin1, ahumanized therapeutic IgG1 monoclonal antibody againstthe HER2/neu proto-oncogen) and rituximab (Rituxan1,chimeric IgG1 monoclonal antibody against CD20) detected

Table 2. Receptors involved in the uptake of opsonized antigens

Name Expression Ligand Function

Fc receptorsFc�RI DC subpopulation IgG, IgG-ICa High-affinity IgG

CD64 bindingFc�RIIA Monocyte-derived IgG-IC ITAM

Fc�RIIB DC # ITIMCD32 Cross priming

inhibitionFc�RIII DC subpopulation IgG-IC ADCCb

CD16Fc�R Interstitial DC IgA-IC Induction of IL-10CD89 production

activationFc"RI Epidermal LC IgE High-affinity IgE

Blood DC bindingsubpopulation Cross priming

activationComplement receptorsC1qR Monocytes, C1q collagen-like Phagocytosis

Macrophages regionCR1 Monocytes, C3b, C4b, iC3b, PhagocytosisCD35 Macrophages MBLd

CR2 B lymphocyte C3d, C3dg B cell co-CD21 FDC stimulationCD21-like IL-7 induced DC EBVe receptorCR3 Monocytes iC3b, C3dg, C3d, Phagocytosis

CD11b-CD18 Macrophages (DC) LPS, fibrinogenICAM-1

CR4 Monocytes iC3b, C3dg, C3d, PhagocytosisCD11c-CD18 Macrophages (DC) fibrinogen

M. tubercolosis

a IC, immune complex. b ADCC, antibody dependent cellular cytotoxicity. c FDC, follicular dendritic cell. d MBL, mannose binding lectin. e EBV,Epstein–Barr virus.

in wild-type nude mice was absent in Fc�R�/� animalsinjected with human breast carcinoma or B-lymphoma cells,respectively. These results indicate that tumor cell-specificIgG type antibodies participate in the stimulation of cellularanti-tumor immune responses by activating DC and bymodifying anti-tumor effector functions.

The high-affinity IgE receptor (Fc"RI) is detectable onmonocytes, DC and LC but lacks the �-chain and is notexpressed constitutively. IgE bound to Fc"RI is efficientlyinternalized to the acidic proteolytic compartments and isdelivered to special MHC class II-rich lysosomal HLA-DMpositive compartments (MIIC). The Fc"RI-dependent sig-naling pathway in DC may be a particularly effective routefor immunization and seems to be a promising target forinterfering with the early steps of allergen presentation(Maurer et al., 1998).

Fc�R (CD89) is expressed on CD14þ interstitial DC ofCD34þ stem cell origin, on monocyte-derived immature DCbut not on LC. CD89 expression is strongly decreased upondifferentiation from monocyte to DC. DC efficiently inter-nalize secretory but not serum IgA without any signs of DCmaturation. This process, however, could not be inhibited bythe anti-CD89 blocking antibody, although it could be blockedby specific sugars or by antibodies specific for the mannosereceptor. These data indicate that immune complexes, com-prising secretory IgA, interact with DC via carbohydratespecific receptors such as the MR (Heystek et al., 2002).

Uptake of apoptotic cells. One of the earliest events ofapoptosis is membrane changes accompanied by the expres-

sion of apoptotic cell-associated membrane proteins(ACAMP; Platt et al., 1998; Larsson et al., 2001). Themembrane pattern of apoptotic cells is recognized byreceptors expressed on phagocytic cells of the innate im-mune system such as tissue DC and macrophages (Table 3).Tissue macrophages play a pivotal role in the clearance ofapoptotic cells without any sign of inflammation mediatedby active anti-inflammatory mechanisms. Rare tissue DC,which reside in nonlymphoid tissues, also act as profes-sional phagocytes and continuously take up apoptotic cells.The receptors, which are involved in the uptake of apoptoticcells by DC, were identified as CD36 and �v�5 integrin incollaboration with the bridging molecule thrombospondin-1(TSP1; Albert et al., 1998a, 2000; Rubartelli et al., 1997).Uptake of apoptotic cells has been shown to be an efficientroute referred as ‘cross presentation’ for loading MHC classI molecules by exogenous viral or tumor antigens. ‘Crosspriming’ of naive T lymphocytes, however, requires simul-taneous danger signals, which results in the activation andmobilization of tissue DC (Plate 4). Lack of activationstimuli leads to the generation of tolerogenic immature orpartially activated DC, which activate regulatory T lympho-cytes by ‘cross tolerance’ (Hackstein et al., 2001). Uptake ofapoptotic cells via phosphatidyl serine (PS) by macropino-cytosis, however, results in efficient presentation of antigensdelivered by the internalized apoptotic cells as well as in‘cross priming’ of cytotoxic T lymphocytes (Hoffmannet al., 2001). The efficient internalization of apoptoticbodies by DC ensures the transfer of a whole set of tumorantigens and thus allows the simultaneous presentation of

Table 3. Receptors involved in the uptake of apoptotic cells

Name Expression Ligand Function

Scavenger receptorsClass-ASR-A I and II Macrophages, DC LPSa (lipid A), LTAb Phagocytosis of bacteria and

apoptotic cellsClass-BSR-B1 Macrophages, DC?, ECc PS, oxidized lipoproteins Phagocytosis, lipid homeostasisCD36 Macrophages, monocytes, DC, EC PS, oxidized lipoproteins Phagocytosis, lipid homeostasisClass-DMacrosialin CD68 Macrophages, EC PS, oxidized lipoproteins Phagocytosis, lipid homeostasisClass-FSREC Macrophages, EC PS, oxidized lipoproteins Phagocytosis, lipid homeostasisNot classifiedSR-PSOX Macrophages, EC PS, oxidized lipoproteins Phagocytosis, lipid homeostasisIntegrinsVitronectin receptor �v�3 Monocytes RGD Phagocytosis of apoptotic cells�v�5 DC RGD Phagocytosis of apoptotic cellsOther receptorsPhosphatidylserine Macrophages, DC Phosphatidylserine (PS) Phagocytosis, lipid homeostasis

receptor (PS-R)ABCA1 transporter Macrophages, DC Lipids Phagocytosis, lipid homeostasisCD14 Macrophages, monocytes LPS Phagocytosis of bacteria and

apoptotic cellsThrombospondin (TSP) Multispan transmembrane TSP Phagocytosis DC inhibition

receptor CD47 protein. Associated with thevitronectin receptor (�v�3)

a LPS, lipopolysacharide. b LTA, lipoteichoic acid. c EC, endothelial cells.

multiple epitopes for both CD4þ and CD8þ T lympho-cytes. In contrast to this route of tumor antigen loading, theamount of soluble tumor antigens may be limited and notsufficient for priming CD8þ T lymphocytes, which requireshigh antigen doses for activation via the exogenous route(Rock et al., 1990). This weak signal could be enhanced byrapid NK cell-mediated tumor cell killing and/or by theproduction of pro-inflammatory cytokines, which sensitizetumor cells for cell death (Ronchetti et al., 1999; Rajnavol-gyi and Lanyi, 2003).

The rapid uptake of apoptotic bodies occurs before DNAfragmentation and may re-utilize the captured DNA forexpression of the coded proteins. Indeed, the uptake ofapoptotic cells was shown to transfer whole or fragmentsof chromosomes into macrophages and DC (Spetz et al.,1999; Holmgren et al., 1999; Bergsmedh et al., 2001).Propagation of the transferred DNA was not observed innormal cells but was shown to occur in cells deficient in p53function (Bergsmedh et al., 2002). Horizontal transfer ofDNA to phagocytes resulted in the intracellular expressionof viral proteins in the nucleus and in the cytoplasm(Holmgren et al., 1999).

Targeting antigen processing and presentationby dendritic cells

DC are up to 1000-fold more efficient in activating resting Tlymphocytes than other professional APC (Bhardwaj et al.,1993). The unique antigen-presenting capacity of matureDC is attributed to the special vesicular system and to thehighly efficient antigen-processing and -presenting machin-ery operating in various DC subtypes (Guermonprez et al.,2002). The antigen-presenting function of DC is highlydependent on microenvironmental factors influencing DCdifferentiation and maturation (Plate 1).

Loading MHC class I molecules. Loading of MHC class Imolecules requires the enzymatic fragmentation of cytosolicproteins by the proteasome. As a result of IFN�-mediatedsignals, different cells replace the standard ‘housekeeping’proteasome by the immunoproteasome, which containsthree novel proteolytic subunits referred to as LMP2,LMP7 and MECL1 (Tanaka and Kasahara, 1998). Theimmunoproteasome acquires altered cleavage specificity,so the spectrum of the generated antigenic peptides mayalso be changed (Groettrup et al., 1995). Monocyte-derivedimmature DC carry approximately equal amounts of thetwo types of proteasome. As a result of maturation, DCup-regulate the expression of the immunoproteasomes(Macagno et al., 1999). Besides IFN�, other cytokineswere shown to induce the expression of different immuno-proteasome subunits in various cell types. TNF� is able toinduce LMP7 but not MECL1 in human endothelial cells(Loukissa et al., 2000). IFN� appears to induce the tran-scription of the LMP2 subunit but not that of the MECL1gene in a renal carcinoma cell line (Hisamatsu et al., 1996).However, the inducible expression of one of the proteasomesubunits may have limited functional consequences, as theintegration of the three subunits into the mature proteasomewas shown to be cooperative (Griffin et al., 1998).

Owing to the distinct enzymatic specificities, some epi-topes can be generated by the immunoproteasome only,while others require processing by the normal proteasome.Thus, the appropriate cleavage of a given antigen by theIFN�-induced immunoproteasome can enhance the presen-tation of some epitopes, while its altered cleavage activitycan also destroy other epitopes (reviewed in Van den Eyndeand Morel, 2001). Proteasomes have at least six active sitesand three distinct cleavage specificities that can be modifiedby inhibitors. Thus the modifying effect on the presentationof a single epitope can range from enhancement to inhibitiondepending on the concentration of the inhibitor (Schwarzet al., 2000). The complete inhibition of proteasome functionby lactacystin, a known inhibitor of proteasome, does notabrogate antigen processing, while the alternative proteasetripeptidyl peptidase II (TPPII) has been shown to be able tosubstitute proteasome function even in the presence of highlactacistyn concentration (Geier et al., 1999).

The renal cell carcinoma ubiquitous antigen RU-1 and themelanoma differentiation antigens MelanA/MART1, gp100/Pmel 17 and tyrosinase were not processed in professionalAPC expressing the immunoproteasome, while MAGE-A3was processed more efficiently (Morel et al., 2000). Theseresults suggest that tumor antigens, which escape appro-priate processing in DC, would escape immune recognitionand thus would be inappropriate for anti-tumor immunother-apy (Van den Eynde and Morel, 2001; Frisan et al., 1998).

DC are also unique in their capacity to direct exogeneousprotein antigens to the MHC class I processing pathway.The phenomenon of ‘cross priming’ was first described byBevan (1976), who observed that CTL responses could beelicited against graft cell antigens, which were presented byautologous MHC class I molecules of the recipient mouse.Cross priming has been considered a major mechanism ofviral and tumor antigen presentation, which could lead topriming of CD8þ cytotoxic T lymphocytes. This mechan-ism ensures immunity against viruses which fail to infectAPC or tumor cells which by themselves are poor APC.Animal studies have demonstrated that DC are sufficient,and probably exclusive in mediating cross-presentation ofdifferent antigens to CD8þ T lymphocytes (Plate 5; DenHaan et al., 2000; Kurts et al., 2001).

The exact mechanism of viral and tumor antigen or DNAtransfer from apoptotic bodies has remained undetermined.In vitro and in vivo studies supported the concept thatunfolded or partially degraded proteins of exogenous pro-teins or apoptotic cells are the major sources of loadedpeptides (Larsson et al., 2001; Albert et al., 1998b). In vivostudies have confirmed the immunogenicity of apoptoticcell-pulsed DC but not of macrophages in cross priming.After pulsing with apoptotic tumor cells only DC showedcross priming and induced tumor-specific CTL (Ronchettiet al., 1999). In addition to antigenic proteins, heat shockproteins (HSP), which act as chaperones for antigenicpeptides and also for empty MHC class I molecules in theER, could be candidates for mediating antigen transfer.

Antigens from multiple extracellular sources could beacquired and presented by DC to MHC class I molecules(Yewdell et al., 1999). Depending on the antigen’s nature,exposure and internalization could also induce DC matura-tion. Necrotic cells, HSP-containing tumor cells, vectorscontaining immunostimulatory sequences (ISS), bacterial

stimulators, dsRNA and CpG nucleotides provide additionalsignals for DC maturation. Cross priming in animal modelsto certain tumor antigens and pathogens has been shown tobe TAP-dependent, suggesting that exogenous antigens canenter the cytoplasm from the endosomal compartements(Huang et al., 1996). However, only small molecules of 3–20 kDa are able to traverse the endosomal membrane, whichsuggests the existence of active transporters, pores or leakyjunctions to mediate cytoplasmic access. Exogenous anti-gens can also be processed in the endosome for access torecycling MHC class I molecules on the same DC. Theadenylate cyclase of Bordetella pertussis binds to CD11b onthe surface of DC and then translocates directly to thecytosol. This allows selective targeting of the MHC class Ipresentation pathway in DC (Guermonprez et al., 2001).Cytotoxic T cell epitopes engineered directly to such vectorselicited strong cytolytic responses against melanoma epi-topes in HLA-A2 transgenic mice.

As a rule, ligand free MHC molecules that happen toreach the cell surface are degraded quickly, and only a fewsurvive, either by reloading their ligand binding site withpeptides present in the extracellular environment or byinternalization in intact form into peptide-rich endosomes.Fluorescence measurements suggested the presence of a fewpercent of empty and still compact MHC class I moleculeson the cell surface, which may acquire peptides fromextracellular sources (Matko et al., 1994). Under in vitroconditions, ligand free MHC class I proteins exhibit a‘molten globule’ state, which was known as an intermediatein their denaturation process (Bouvier and Wiley, 1998;Simon et al., 2000). This conformational state is much lesscompact than that of the native protein and may interactdifferently with other proteins such as chaperones (Simonet al., 2000). Based on these results, the nature of the antigenand the accompanying activation signal in combination withthe available cytokines can modify antigen processing andpresentation of DC and offers possibilities for targeting theMHC class I pathway. These factors may act in concert anddetermine the outcome of T lymphocyte activation.

Loading MHC class II molecules. Unlike other APC, DCalter their capacity to present peptides via MHC class IImolecules during differentiation, which points to a complex,and multilevel regulation of antigen presentation by matureDC. Antigens acquired from exogenous sources are pro-cessed within specialized MHC class II–rich compartments(MIIC) detected in DC as a result of activation and matura-tion, which induces the transport of peptide-loaded MHCclass II molecules to the plasma membrane. During thematuration process DC translocate MHC class II-peptidecomplexes together with the CD86 costimulatory moleculeto the cell surface, which results in the efficient activation ofspecific CD4þ helper T lymphocytes (Inaba et al., 2000;Turley et al., 2000).

The specific proteases involved in exogeneous antigenprocessing include a wide range of proteases such ascathepsins and asparaginyl endopeptidases. Most of theseacidic proteases have broad substrate specificity. CathepsinH and C act as amino-peptidases whereas cathepsin B and Zfunction mainly as carboxy-exopeptidases. Antigen proces-sing is tightly regulated in maturing DC by controllingprotease inhibitors through inflammatory mediators (Watts,

2001). Immature DC with high endocytic capacity areineffective in loading MHC class II molecules with peptidesprocessed from the internalized proteins unless they receivea maturation signal. This process is controlled bythe regulation of cathepsin S expression, which is respon-sible for the degradation of the invariant chain and thus forthe availability of empty MHC class II peptide binding clefts(den Haan et al., 2000).

Ligand-free MHC class II molecules were detected on thesurface of immature DC and of B lymphodytes usingconformation-dependent monoclonal antibodies. These mo-lecules were shown to be functional both in antigen bindingand in subsequent T-cell activation (Santambrogio et al.,1999a). HLA-DM, a key chaperone keeping MHC class IImolecules in a peptide receptive form in the endosome andwhich facilitates the exchange of the invariant chain-derivedCLIP and antigenic peptides was also detected on thesurface of DC (Arndt et al., 2000). Based on these results,a local extracellular antigen processing pathway was sug-gested, which is mediated by membrane bound and/orsecreted proteases in the environment of DC, which couldbe involved in the loading of ligand-free MHC class IImolecules (Santambrogio et al., 1999b).

Certain cytokines such as IL-10 are able to modify theprocessing of endocytosed antigens by modulating cathe-psin activity and MHC class II trafficking via regulation ofinvariant chain proteolysis in monocytes and DC(Koppelman et al., 1997; Fiebiger et al., 2001). IL-6 doesnot induce conventional maturation of immature DC but isable to alter the pH of early endosomal compartments. As aresult, DC exposed to IL-6 were able to prime T lympho-cytes specific for previously cryptic antigenic determinants(Drakesmith et al., 1998).

Targeting activation and maturation of dendritic cells

An important step of in vitro DC preparation is the deliveryof the appropriate activation signal, which ensures themobilization and maturation of DC. In clinical settingsthis in vitro provided signal should not precede the optimalloading of immature DC since maturation down-regulatesantigen uptake. The in vivo situation also requires vaccinecomponents, which facilitate the recruitment of DC to thevaccination site.

Maturation in the presence of various cytokines. A widerange of stimuli have been shown to activate DC, whichinclude various cytokines, microbial compounds, moleculesarising from cellular damage, from interaction with acti-vated T lymphocytes, which can be substituted by solubleCD40 ligand. Despite the variability of differentiation andmaturation protocols, in vitro generated monocyte-derivedDC share phenotypic characteristics and functional activ-ities. These include the up-regulation of co-stimulatorymolecules, decrease of phagocytic activity, increased abilityto induce T cell proliferation in mixed lymphocyte reactionsand changes of migratory properties of DC. Accumulatingdata suggest that the stimulus used for DC differentation andactivation affects the functional properties of mature DC.Microarray and SAGE studies indicated fundamental differ-ences in gene expression profiles of DC activated under

different conditions (Hashimoto et al., 2000). The simplecomposition of culture medium determined the capacity ofDC for tuning T cell activation towards Th1 or Th2 direc-tions (Chang et al., 2000). Timing of activation stimuli uponDC differentation of mouse bone marrow-derived DC influ-enced the generation and ratio of IL-12 or IL-10 producingDC (Jiang et al., 2002). In a similar system LPS and CD40crosslinking induced different cytokine expression profilesof DC-derived cytokines as both stimuli resulted in in-creased TNF�, IL-6, IL-12p40 and IL-15 mRNA levels,but only LPS up-regulated IL-1�, IL-1� and IL-12p35expression. In accordance with their cytokine pattern LPS-activated DC were more potent inducers of Th1-type re-sponses as compared with DC activated by CD40 cross-linking (Morelli et al., 2001). These studies indicated thatmodifying culture conditions of DC may result in subtlechanges in effector functions. This type of functional flex-ibility has substantial impact on DC-based immunotherapy.

Various cytokines were shown to influence DC activationproviding invaluable tools for fine tuning DC activities.TNF�, IL-1�, IFN� and IL-12 were shown to induce DC1with the capacity to elicit strong Th1 responses. MCP-1treatment of DC, however, selectively inhibited Th1 re-sponses without affecting polarization to Th2 (Omataet al., 2002). Human peripheral blood DC, activated withthymic stromal lymphopoietin (TSLP), primed naive Thcells towards a unique cytokine expressing profile. TSLPdown-regulated IL-10 and IFN� production in T cells ascompared with other activation stimuli, while these cellsproduced high amounts of Th2 cytokines together withTNF� (Soumelis et al., 2002). TGF� was shown to suppressthe activation of some types of DC (Geissmann et al., 1999)and IL-10 to inhibit DC maturation, which resulted in thedown-regulation of both Th1 and Th2 immune responses(Haase et al., 2002).

Many cytokines involved in the activation or suppressionof DC effector functions, such as TNF�, IL-1�, IL-6, IL-12or IL-10, are produced by the DC themselves and thus act inan autocrine manner. Other cytokines, such as IL-2, wereshown to modulate the activity of DC. Uptake of bacteriawas able to induce early but transient production of IL-2,which had an adjuvant effect on DC-mediated T cellpriming (Granucci et al., 2002). The T cell stimulatorycapacity of IL-2 again may act on the DC itself and/or onother cells of innate and adaptive immunity. IFN� is a potentadjuvant of Th1 responses and acts directly on monocyte-derived DC. In combination with monocyte conditionedmedium or LPS, IFN� increased the release of IL-12,TNF� and the Th1 recruiting CXCL10 (IP-10) chemokine.The production of IL-10 and CCL17 (TARC) chemokine,both supporting Th2-deviated or suppressed immune re-sponses, were downregulated (Corinti et al., 2002). In theseexperiments a novel protein delivery system was utilizedusing biotinylated HIV Tat protein coupled to biotinylatederythrocytes by avidin (Magnani et al., 1994).

Other factors or drugs, which modify DC maturation,may also influence DC-mediated T cell activation. Imma-ture or partially mature DC, which express co-stimulatorymolecules and MHC II-peptide complexes but lack cytokineproduction, were shown to down-regulate T cell-mediatedimmune responses by inducing T cell anergy and regulatoryT cell differentiation, respectively (Lutz and Schuler, 2002).

Tumor cells, genetically modified by various cytokines,were tested in mice for augmenting anti-tumor immunityand the beneficial effect of GM-CSF and IL-12 was con-firmed in several experiments (Schmidt-Wolf and Schmidt-Wolf, 1995). To overcome their short half-life and their invivo side effects when given at high doses or systematically,cytokines can be encapsulated in microspheres. The cyto-kine genes can also be transferred in vivo. Similar totraditional adjuvants, an alternative approach would be touse agents that induce cytokine production at the site ofinjection. Genes that code for microbial molecules capableof triggering cytokine release are promising candidates forproducing such adjuvants with longer in vivo half-life thancytokines. One example is LEIF (Leishmania elongationinitiation factor), which up-regulates the expression of theB7 co-stimulatory molecules on APC and stimulates theproduction of IL-12 from APC and IFN� release from NKcells (Probst et al., 1997).

Modifying the activation state of dendritic cells bytargeting cell surface molecules. Immature DC, whichare localized to non-lymphoid tissues, can be activated byexogenous and endogenous danger signals, such as patho-genic products or inflammatory cytokines (Plate 4; Ulevitch,2000; Aderem and Ulevitch, 2000; Schnurr et al., 2000;Marriott et al., 1999). The maturation signal is essential indetermining the immunogenic or tolerogenic capacity of theDC (Larsson et al., 2001). Apoptotic cell internalizationby DC also depends on the presence of additional ‘dangersignals’, which regulate DC differentiation (Hawiger et al.,2001).

Negative signals inhibiting DC maturation. In addition tomodulation of DC phenotype by cytokines, various surfacereceptors expressed by immature DC were shown to influ-ence DC maturation. The adhesion molecule E-cadherin,which is involved in homophylic interactions of Langerhanscells and tissue epithelial cells, transduces negative signalsto LC and inhibits their maturation. Activation of LC byinflammatory cytokines results in the down-regulation of E-cadherin expression and termination of E-cadherin-mediated suppression of DC activation (Riedl et al., 2000).

Uptake of normal tissue cells or apoptotic tumor cellsalone does not induce DC maturation (Sauter et al., 2000;Gallucci et al., 1999). Internalization of apoptotic cells bythe CD14 receptor complex expressed by macrophages butnot by DC does not result in inflammation either and is ableto inhibit signaling via LPS (Byrne and Reen, 2002). Tollreceptors also bind LPS and mediate danger signals for DC(Table 3). The ‘alternative’ activation of macrophages byIL-4 and IL-13 results in IL-10 and TGF�1 production(Gordon, 2003). IL-10 favors the generation of tolerogenicDC (Jonuleit et al., 2001) and the induction of regulatory Tcells is also supported by the immunosuppressive cytokineTGF� (Savill et al., 2002).

Ligation of CD36 molecules in DC, which are involved inbinding of apoptotic cells, resulted in decreased IL-12production and promoted the expression of IL-10 (Urbanet al., 2001). Uptake of apoptotic cells by immature DC viaCD36 inhibits maturation of DC and results in immunesuppression (Urban et al., 2001). CD36 is also targeted byPlasmodium falciparum-infected erythrocytes and leads to

the down-regulation of IL-12 production. The inhibitoryeffect of CD36 ligation is suggested as one of the majorimmune evasion mechanisms in patients suffering frommalaria (Urban et al., 1999).

Thrombospondin (TSP) is transiently expressed at highconcentrations in damaged and inflamed tissues and haspotent anti-inflammatory capacity (Bornstein, 1992). Thethrombospondin (TSP) receptor CD47, also referred asintegrin-associated protein, is a multispan transmembraneprotein associated with the vitronectin receptor �v�3. CD47is expressed on human monocyte-derived DC in a function-ally competent form. However, ligation of CD47 inhibitedthe phenotypic and functional changes associated with thematuration of DC as well as the secretion of pro-inflamma-tory cytokines without affecting the uptake of apoptoticbodies (Demeure et al., 2000).

CD83 belongs to the Ig superfamily and is considered as aspecific marker of mature DC. The soluble extracellulardomain of CD83, present also in serum, was recently shownto exert T cell inhibitory activity. In addition to this effect,soluble CD83 also interfered with DC maturation andresulted in the down-regulation of CD83 itself and also ofthe CD80 co-stimulatory molecule (Lechmann et al., 2002).

Positive signals supporting DC maturation. Targetingantigen-antibody complexes by Fc- and complement recep-tor-mediated binding and internalization may have opposingeffects on the antigen presenting and T-cell activatingcapacity of various APC (Mosser and Karp, 1999). Bindingof immune complexes to Fc- and complement-receptors wasshown to down regulate IL-12 production in macrophagesand to suppress Th1 responses (Mosser, 2003). In contrast tomacrophages, internalization of immune complexes viavarious FcR of DC not only targets MHC class I and classII molecules but also delivers activation signals. The Fc�R-mediated antigen internalization pathway confers a matura-tion signal for immature DC and sensitizes them for primingboth CD4þ and CD8þ T lymphocytes (Regnault et al.,1999). Aggregation of Fc"RI on monocytes results in theproduction of IL-10, prevents DC differentiation in thepresence of IL-4 and GM-CSF and results in the generationof macrophage-like cells with low level of CD1a (Novaket al., 2001). Ligation of Fc"RI of primary human mono-cytes and DC by polyvalent antigens results in NF-�Bactivation and the release of TNF� and monocyte chemoat-tractant protein-1 (MCP-1), indicating the potency of Fc"RIto control inflammatory reactions (Kraft et al., 2002). Cross-linking of the Fc�R receptor induces internalization, IL-10production, up-regulation of MHC class II and CD86costimulatory molecules and results in increased allostimu-latory activity (Geissmann et al., 2001).

Synthetic oligonucleotides containing unmethylated CpGmotifs activate professional APC and also lymphocytes andNK cells to release various cytokines and chemokines inmice. CpG act via Toll receptor-9 and initiate intracellularsignaling. In the human system two distinct CpG motifshave been identified. One of these motifs stimulates DC forIL-6 secretion and B lymphocytes for IgM production, whilethe other one stimulates NK cells for IFN� release(Verthelyi et al., 2001).

The high mobility group 1 (HMGB1) protein acts as anarchitectural DNA chromatin-binding factor, which

promotes the binding of nuclear factors by DNA bending.Chromatin binding activity of the protein is highly depen-dent on the viability of the cell and differs in apoptotic andnecrotic cells. In apoptotic cells HMGB1 is tightly bound tochromatin and its release is induced only if de-acetylation isprevented. These results demonstrate that necrotic but notapoptotic cells release HMGB1, described as an inflamma-tory protein secreted by activated monocytes and macro-phages (Scaffidi et al., 2002).

Danger signals can be delivered by the apoptotic cellitself or by stimuli derived from pathogens or from in-flammed or stressed tissues (Plate 4). Besides microbialLPS, glucanes, CpG oligonucleotides (ODN), lipids orglycolipids, the metabolic products of altered, damaged,senescent or dying cells are known mediators of DCactivation while the contact with necrotic tumor cells alsoresults in DC activation (Sauter et al., 2000; Gallucci et al.,1999). Some of these stimuli act directly on DC viaToll receptors or indirectly through other cells of the innateimmune system, such as NK-cells. IFN� is a potentactivator of macrophages and induces the production ofpro-inflammatory cytokines such as IL-1�, IL-6 and TNF�,GM-CSF and IL-8 involved in the recruitment of neutrophilgranulocytes. Necrosis, which may occur at the late stage oftumor development, induces local inflammation and thusfavors the generation of tumor-specific immunity.High tumor burden and the immune suppressive microen-vironment of the tumor tissue, however, may limit thefunctional efficacy of effector T cells even in necrotizingtumor tissues.

HSP are stress-induced intracellular proteins, which actas endogenous danger signals via activation of the innateimmune system (reviewed in Wallin et al., 2002). Variousreceptors can mediate stimulatory signals by HSP, such asCD91, CD14 and Toll receptor-2 and -4, all expressed onprofessional APC. The uptake of HSP-peptide complexes is,however, restricted to CD91, which binds gp96, HSP70 andHSP90 (Ohashi et al., 2000; Binder et al., 2000; Asea et al.,2000). HSP, isolated from tumor cells, were shown to carryfragments of tumor antigens and necrotic but not apoptoticcells were found to release HSP and activate DC (Basu et al.,2000). The immunogenicity of tumor cell lines was relatedto their HSP70 content and tumor antigens coupled withHSP were demonstrated to be efficient anti-tumor vaccines(Melcher et al., 1998).

In addition to in vitro manipulation of DC phenotype andfunctions, effector activities of ex vivo manipulated re-injected DC may further be modified in vaccinated subjects.Monocyte-derived DC, activated with the inflammatorycocktail of TNF�, IL-1�, IL-6 and PGE2 did not producehigh amounts of IL-12; however, these cells were able toinduce strong Th1 responses following subcutaneous injec-tion. This result indicates that DC maturation is continued asa result of interaction with the tissue environment (Schuler-Thurner et al., 2002).

We can conclude that DC maturation is highly sensitive toin vitro and in vivo conditions and micro-environmentalstimuli. In view of the tolerogenic and immunogenic poten-tial of manipulated DC and their capacity to polarize helperT-lymphocytes either to Th1 or Th2 effectors, DC emerge asprimary targets for both positive and negative immuneregulation.

Targeting dendritic cell migration and homing

Mobilization of tissue DC at the right time is critical inpriming cellular immune responses against viruses andtumors. Following microbial infection or tissue injury,peripheral blood monocytes migrate to sites of inflammationand differentiate to macrophages and DC. Together withtissue resident immature interstitial DC, monocyte-derivedDC participate in antigen uptake and in transport of inter-nalized material into lymphoid tissues (Plate 4). Migrationto sites of inflammation is regulated by various chemokinesproduced by tissue resident macrophages and by the DCthemselves (Luster, 2002).

In response to activation stimuli, the expression of che-mokine receptors on the surface of DC changes and the cellsbecome responsive to chemokines, which are produced inlymphoid tissues such as CCL19 (MIP-3�) and CCL21(SLC; Sallusto and Lanzavecchia, 2000). Intra-tumoralinjection of a MIP-3�-adenovirus vector attracted immatureDC and inhibited tumor growth in mouse syngeneic tumormodels (Fushimi et al., 2000). Immature epidermal Langer-hans cells could be entrapped by the Langerhanscell-attracting macrophage inflammatory protein (MIP-3�)chemokine gradient and then the captured DC could beloaded with antigen in situ at the site of the tumor(Kumamoto et al., 2002).

Migration of DC from non-lymphoid tissues to lymphoidorgans and within lymphoid microenvironments dependsnot only on chemokine receptor expression and chemokinegradients, but also on the remodeling of extracellular matrixmediated by special enzymes attacking cell surface recep-tors expressed in the membrane of DC. Disintegrin metal-loproteinases act as adhesion molecules mediated by theirC-terminal disintegrin domain and have a potential cleavagefunction through the zinc-dependent metalloprotease do-main. This type of enzyme was also shown to be involved inthe cleavage of membrane-associated cytokines such TNF�(Black et al., 1997), FasL (Kayagaki et al., 1995), TGF�,GM-CSF-� subunit (Prevost et al., 2002) and the homingreceptor L-selectin (Bennett et al., 1996). Two newlyidentified members of this protein family are specific forDC and are referred to as decysin and metalloprotease anddisintegrin dendritic antigen maker (MADDAM) or metal-loprotease and disintegrin family19 (ADAM19). Decysin isexpressed in mature human DC and in tonsillarCD11cþCD4þ germinal center residing DC (Muelleret al., 1997). Mouse decysin was detected in folliculardendritic cells (FDC), tingible body macrophages and alsoin subcapsular macrophages, which upon immunizationmigrate to germinal centers and differentiate to matureDC (Mueller et al., 2001). MADDAM was identified as adifferentiation and activation marker for human DC, whichcan distinguish between macrophages and DC (Fritscheet al., 2000).

DC migration is also regulated by other mechanisms. Theleukotriene C4 transporter function of the multidrug resis-tance associated protein-1 (MRP-1) seems to be essential inthe in vivo mobilization of mouse DC from the epidermis.Inhibition of MRP-1 prevented in vitro migration of humanmonocyte derived DC in response to CCL19 in chemotaxischamber (Robbiani et al., 2000). Another multidrug resis-tance protein, P-glycoprotein (MDR-1) was also shown to

confer similar activity in DC migration demonstrated by thestrong promotion of human dermal DC trafficking viaafferent lymphatic vessels (Randolph et al., 1998). Humanmonocyte-derived DC differentiated in the presence of GM-CSFþ IL-4 was shown to express MRP-1 but not the P-glycoprotein (Laupeze et al., 2001).

Adhesion receptors, such as CD62L, LFA-1 and DC-SIGN, which participate in the binding of DC to endothelialcells or E-cadherin binding to epithelial cells may also directthe routes of DC trafficking (Martin et al., 2002). Metallo-protease activity or endogenous enzyme inhibitors may alsobe targets for modulating the migration of DC (Osman et al.,2002).

Many pathogens utilize the unique features of DC,especially their migratory capacity, for spreading. MucosalLC play an essential role in disseminating viruses or otherpathogens from mucosal surfaces to draining lymph nodes(reviewed in Guermonprez et al., 2002).

Proper migration and homing of antigen-pulsed DC invaccinated tumor-bearing individuals is an essential require-ment for the therapeutical effect. The route of DC-basedimmunization strongly influenced the magnitude and thequality of the tumor-specific immune response. In a clinicalsetting, recombinant tumor antigen-pulsed DC were admi-nistered by intravenous, intradermal or intra-lymphatic in-jections into patients with metastatic prostate cancer.Activated DC were able to prime antigen-specific T cellresponses regardless of the route of injection, whereas IFN�production was induced by intradermal or intra-lymphaticinjections only, whereas antigen-specific antibody produc-tion was more frequent following intravenous immunizationas compared to intradermal or intralymphatic inoculations(Fong et al., 2001).

Transcutaneous immunization targets skin resident LC.This approach was used to deliver antigen in combinationwith the heat labile enterotoxin (LT) of E. coli. A phase Iclinical trial demonstrated that in contrast to oral or nasaladministration the cutaneous route avoided toxicity of LTand elicited both IgG and IgA type antibody productionagainst an E. coli colonization factor used as a modelantigen (Hammond et al., 2001).

Targeting dendritic cells by exosomes

Exosomes are vesicles of 30–100 nm in diameter, which aresecreted by various cells in culture as a consequence offusion of multivesicular late endosomes/lysosomes with theplasma membrane (Thery et al., 2002a). Exocytosis ofmultivesicular bodies (MVB) is mediated by inverse bud-ding, which correlates with the inversion of the transmem-brane partition of the lipid phosphatidylserine. MVB hasbeen described in various cell types mostly of hematopoieticorigin among them dendritic cells (Zitvogel et al., 1998).

Analysis of the protein composition of exosomes derivedform various cells revealed the presence of commonproteins, which define exosomes as a bona fide secretedsubcellular compartment, as well as the presence of somecell-type-specific proteins, which mediate cell-type-depen-dent functions. Most importantly, exosomes exhibit a dif-ferent array of proteins as compared with that of apoptoticblebs (Thery et al., 2001). Proteins that have been identified

in murine and human DC-derived exosomes are summar-ized in Table 4 (Thery et al., 1999, 2001; Lamparski et al.,2002; Clayton et al., 2001, 2003; Zitvogel et al., 1998).Exosomes were shown to be resistant to extracellulardegradation by the complement system, which was ensuredby the GPI-anchored complement regulators CD55 (delayaccelerating factor, DAF) and CD59 (Protektin) on exo-somes (Clayton et al., 2003).

The overall analysis of exosome-associated proteinsrevealed that they carry proteins of cytosolic origin whichderive from the plasma or endosomal membranes. Theseresults also suggest that these vesicles overexpress mole-cules and receptors potentially involved in targeting anti-gens to professional APCs. There are several potentialmechanisms of how exosomes can interact with a targetcell, which include binding to the cell surface, fusion withthe plasma membrane or uptake of vesicles by endocytosis.DC-derived exosomes were shown to express molecules thatare good candidates for such docking functions. The se-creted glycoprotein milk fat globule-EGF-factor 8 (MFG-E8) is produced by macrophages and is expressed also inexosomes. It is able to bind to �v�3 integrin via its RGDmotif (Albert et al., 2000; Hanayama et al., 2002).

The uptake and/or the presentation of exosome-asso-ciated ovalbumin by DC requires functional CD91 receptors(Skokos et al., 2003). As a cytosolic protein, hsc70 isexpected to be present in the lumen of exosomes (Hanayamaet al., 2002); however, it has also been detected on thesurface of exosomes derived from macrophages and DC(Arnold-Schild et al., 1999). Thus, it is able to mediateinternalization by the CD91 receptor.

Exosomes can activate naive T lymphocytes in vivo andin vitro. Exosomes loaded with specific antigens can activateCD4þ T cell clones in vitro, albeit 10–20 times lessefficiently than B lymphocytes (Raposo et al., 1996). Invitro stimulation required two distinct DC populations, onefor producing exosomes bearing intact antigens or MHCclass II-peptide complexes, the other one for taking up theseexosomes for re-processing or for acquiring MHC class II-peptide complexes from exosomes to activate helper Tlymphocytes (Thery et al., 2002b). Exosomes released byDC, which had been pulsed with acid-eluted tumor-derivedpeptides, also activated tumor-specific CD8þ T cells invitro; however, the stimulation by exosomes was lessefficient than by intact cells displaying peptide-loadedMHC class I molecules (Zitvogel et al., 1998).

Table 4. Molecular composition of dendritic cell derived exosomes

Human Mouse Functions

GPI-anchored proteinsCD55/DAF Regulation of complement activationCD59/MIRL Protection from complement mediated lysis/signalingCD58/LFA-3 (GPI-isoform) Adhesion/co-stimulationImmunoglobulin-supergenefamily

MHC class II molecule MHC class II molecule Antigen presentationMHC class I molecule MHC class I molecule Antigen presentationCD1a Antigen presentationCD1b Antigen presentationCD1c Antigen presentationCD1d Antigen presentationCD86/B7.2 CD86/B7.2 Co-stimulationCD54/ICAM-1 Adhesion to target cellsTetraspan membrane protein familyCD9 CD9 Signal transduction/adhesion/complex formation

with MHC and integrinsCD63/Lamp-3 CD63/Lamp-3 Signal transduction/adhesion/complex formation

with MHC and integrinsCD81 Signal transduction/adhesion/complex formation

with MHC and integrinsCD82 CD82 Signal transduction/adhesion/complex formationCytosolic proteins

hsc73, hsp84 Antigen-derived peptide bindingGi2 �-subunit Signal transductionAnnexin I, II, IV, V, VII Membrane fusion

OthersTfR Iron transport

CD11b (Mac-1/integrin CD11b (Mac-1/integrin �-chain) Adhesion to target cells�-chain)

CD11c (�X subunit Stimulates phagocytosisof CR4, binds fibrinogen)

MFG-E8/lactadherin Adhesion to target cells

The low efficiency of exosome-induced stimulation of T-lymphocytes in vitro compared with the dramatic effect ofDC-derived exosomes in vivo suggests that exosomes do notinteract directly with CTL. They may instead work astransport vehicles for immunogenic antigens from immatureDC located in the periphery to other DC, which getsensitized for T cell stimulation (Denzer et al., 2000).

Mast cell-derived exosomes are also able to inducematuration and functional activation of DC through cross-presentation of antigen to T cells (Tkaczyk et al., 1996).Mast cell-derived HSP-containing exosomes were shown toact as direct inducers of DC maturation being responsiblefor both the mitogenic activity of exosomes and the highimmunogenic potential of exosome-associated antigens(Tkaczyk et al., 1996).

Tumor-derived exosomes emerged as a novel source oftumor rejection antigens and an efficient method of DCloading. They can also be useful for the characterization ofimmunologically relevant tumor antigens and for autolo-gous or allogeneic cancer immunotherapy. Indeed, tumorcell-derived exosomes were shown to concentrate a set ofshared tumor-rejection antigens such as Mart-1, which wasefficiently taken up and cross-presented for specific T cellclones by MHC-I molecules on human DC (Wolfers et al.,2001). Regardless of their putative physiological functions,exosomes have successfully been used in preclinical studiesconducted in mouse and human systems and a Phase Iclinical trial with melanoma patients is currently ongoing(Hammond et al., 2001). Tolerosomes derived from smallintestinal epithelial cells represent another example ofexosome like structures (Karlsson et al., 2001). Tolerosomesisolated from serum shortly after antigen feeding or from invitro-pulsed intestinal epithelial cells are capable of indu-cing antigen-specific tolerance in naive recipient animals.

CONCLUDING REMARKS

Dendritic cells are rare but multifunctional cells, which actas sentinels and sensors in non-lymphoid tissues. Uponactivation, induced by inflammation or pathogens, theymigrate to peripheral lymphoid tissues, where they function

as highly professional antigen-presenting cells. DC repre-sent a diverse and flexible cell population and their physio-logical functions in maintaining self-tolerance or primingvarious cellular immune responses can be modulated atmultiple levels. The unique and diverse properties of DCoffer multiple possibilities for targeting their activity forbetter T lymphocyte activation or for more potent regulationof various immune responses. One of the most promisinguses of properly educated DC is their application as adju-vants, delivery systems or vehicles for cancer vaccines inhuman therapeutical settings. These individual immunother-apeutic approaches either use ex vivo educated antigen-loaded DC or target DC in vivo with antigens, cytokines orchemokines. DC also become promising targets of genetherapy for eliciting anti-viral or anti-tumor immune re-sponses. Owing to their PPR, ACAMP, Toll and cytokinereceptors, DC act as potent effectors of innate immunity,whereas their antigen-presenting, adhesion and co-stimula-tory receptors facilitate collaboration with cells of acquiredimmunity. Activation of helper T lymphocytes responsiblefor inflammatory or allergic reactions and the priming ofcytotoxic effector T cells is directed by DC, which transducetissue-derived stimuli to activation signals for T lympho-cytes. Interestingly enough, DC are also major regulators ofimmunological tolerance, tissue rejection, autoimmunityand allergy. Manipulation of DC polarization in a tolero-genic or inflammatory direction facilitates their migrationand homing to relevant tissues; this provides multiplepossibilities for regulating DC functions and consequentlyimmune responses. The promising results obtained withtargeting DC functions summarized in this article are justthe beginning of a complex interdisciplinary research activ-ity and are likely to lead to more specific and efficient exvivo and/or in vivo DC targeting strategies.

Acknowledgements

The work was supported by grants of the National Science Foundation ofHungary (OTKA T043420, TS044798) and from the R&D projects NKFP-00088/2001 and BIO/00032/2001. We thank Arpad Lanyi for criticalreviewing the manuscript.

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Targeting dendritic cells for priming cellular immune responses

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